Abstract
Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
Highlights
Haloalkane dehalogenases are microbial enzymes acting on haloorganic compounds
The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins
Seven different amino acid residues Ala, Cys, Phe, Gly, Lys, Thr, and Trp were introduced in the position 177 of LinB by site-directed mutagenesis to investigate the role of Leu177 on catalytic efficiency and substrate specificity
Summary
Haloalkane dehalogenases are microbial enzymes acting on haloorganic compounds. The enzymes cleaves the carbon-halogen bond and replaces a halogen with a hydroxyl group from a water molecule [1]. Engineered enzymes can be used in biotechnology applications, such as detoxification of environmental pollutants and bioorganic synthesis. Such technologies are already in use [14] or are under development [2, 15, 16]. A comparison of these structures revealed that are the size, shape, and physicochemical properties of the active site important determinants of specificity and the size and shape of the entrance tunnel [30]. In this study we attempted to modify the specificity of haloalkane dehalogenase LinB by engineering of the tunnel connecting the protein surface with its active site cavity. Amino acid residue Leu177, which is positioned in the tunnel opening (see Fig. 1), was replaced, and the effect of the mutation on enzyme activity and specificity was studied
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
